Development of new in situ hybridization method using freeze transfer.

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In situ hybridization (ISH) is an useful technique to localize gene expression in a tissue section. Its time consuming and complex procedure requires great skill of researchers. In the present study, we developed simpler and sensitive ISH method by mean of freeze transfer technique.Limbs and heads, obtained from 14-day embryonic mouse, were frozen and embedded in OCT compound. Frozen sections (8mum thick) were mounted directly onto the surface of nitrocellulose membrane and were quickly dried by hair drier. Membranes were then treated with 10mug/ml protenase K for 10-40- min, refixed with 4% PFA,and hybridized with digoxigenin-labelled type I,II,X collagen, and qggrecan riboprobes. In a parallel experiment, conventional ISH were carried out with same tissue sections and probes.In all experiments, we obtained higher signal and lower back-ground by our new ISH.This technique was superior to a conventional ISH in following points. Once the tissue section were mounted on the membrane, tissue sections were not peeled off from the membrane even after 70ﾟC hybridization and 70ﾟC wash. We could omit acetylation and RNase treatment step which is usually necessary to reduce back-ground. In addition, our new ISH required only 1/10-1/100 of probe amount which was used in a conventional ISH.Therefore our new ISH is technically simpler and more sensitive than conventional ISH.